Novel tetragalnac containing conjugates and methods for delivery of oligonucleotides

ABSTRACT

Disclosed herein is a modular composition comprising 1) an oligonucleotide; 2) one or more tetraGalNAc ligands of Formula (I), which may be the same or different; optionally, 3) one or more linkers, which may be the same or different; and optionally, 4) one or more targeting ligands, solubilizing agents, pharmacokinetics enhancing agents, lipids, and/or masking agents.

BACKGROUND OF THE INVENTION

Scientific efforts focused on the delivery of oligonucleotidessystemically for therapeutic purposes are ongoing. Three highlightedapproaches to oligonucleotide delivery include 1) lipid nanoparticle(LNP) encapsulation, 2) polymer conjugation and 3) single chemicalconjugation. Single chemical conjugation typically employs a targetingligand or a lipid or a solubilizing group or an endosomolytic peptide ora cell penetrating peptide and/or a combination of two or all fourattached to an oligonucleotide. Linkers may be present in the conjugateas well as other functionalities. Single chemical conjugates are knownand attachment of the oligonucleotide occurs either at the 5′- or 3′-endof the oligonucleotide, at both ends, or internally. See WO2005/041859,WO2008/036825, and WO2009/126933. Considerable amount of literatureevidence supports the hypothesis that the major hurdles foroligonucleotide delivery are cell uptake and endosomal escape. Thereremains a need for additional single chemical conjugates that canprovide effective delivery efficiency, cell uptake and/or endosomalescape.

SUMMARY OF THE INVENTION

Single chemical conjugates comprising tetraGalNAc ligands disclosedherein have surprising properties of improved delivery efficiency, celluptake and/or endosomal escape.

In one embodiment, a modular composition disclosed herein comprises: 1)a single stranded or double stranded oligonucleotide; 2) one or moretetraGalNAc ligands of Formula (I), which may be the same or different:

wherein X is —O—, —S—, —R¹R²— or —NR¹—, wherein R¹ and R² are eachindependently selected from the group consisting of hydrogen andC1-6alkyl; n is 1, 2, 3, or 4; and the bond with “

” indicates point of attachment; optionally, 3) one or more linkers,which may be the same or different; and optionally, 4) one or moretargeting ligands, solubilizing agents, pharmacokinetics enhancingagents, lipids, and/or masking agents. In one embodiment, R¹ and R² areeach independently selected from the group consisting of hydrogen,methyl and ethyl. In another embodiment, R¹ and R² are each hydrogen.

In one embodiment, the tetraGalNAc ligand has Formula (II) wherein X,R¹, R² and n are as defined above. In another embodiment, thetetraGalNAc ligand has Formula (III) wherein X, R¹, R² and n are asdefined above:

In another embodiment, a modular composition comprises: 1) a singlestranded or double stranded oligonucleotide; 2) 1-8 tetraGalNAc ligandsof Formula (I), (II) or (III), which may be the same or different,wherein X is —O—, —S—, —H₂— or —NH—; and n is 1, 2, 3, or 4; 3) 1-24linkers, which may be the same or different; and optionally, 4) 1-8targeting ligands, solubilizing agents, pharmacokinetics enhancingagents, lipids, and/or masking agents.

In another embodiment, a modular composition comprises: 1) a singlestranded or double stranded siRNA; 2) 1-8 tetraGalNAc ligands of Formula(I), (II) or (III), which may be the same or different, wherein X is—O—, —S—, —H₂— or —NH—; and n is 1, 2, 3, or 4; 3) 1-16 linkers, whichmay be the same or different; and optionally, 4) 1-8 targeting ligands,solubilizing agents, pharmacokinetics enhancing agents, lipids, and/ormasking agents.

In one subset of the above embodiments, the linkers are attached to theoligonucleotide or siRNA at different 2′-positions of the ribose ringsand/or at different terminal 3′ and/or 5′-positions of theoligonucleotide or siRNA.

In another subset of the above embodiments, the tetraGalNAc ligands areattached to the oligonucleotide or siRNA optionally via linkers.

In another subset of the above embodiments, the tetraGalNAc ligands areattached to the oligonucleotide or siRNA at different 2′-positions ofthe ribose rings and/or at different terminal 3′ and/or 5′-positions ofthe oligonucleotide or siRNA; and the tetraGalNAc ligands are attachedto the oligonucleotide or siRNA optionally via linkers.

In another subset of the above embodiments, X of Formula (I), (II) or(III), is —O—, —S—, or —H₂—; and n is 1, 2 or 3.

In another subset of the above embodiments, X of Formula (I), (II) or(III), is —O— or —H₂— and n is 1 or 2.

In another subset of the above embodiments, X of Formula (I), (II) or(III), is —O— and n is 1 or 2.

In another subset of the above embodiments, X of Formula (I), (II) or(III), is —CH₂— and n is 1 or 2.

In another subset of the above embodiments, the composition comprises1-6 tetraGalNAc ligands, or more specifically, 1-4 tetraGalNAc ligands,which may be the same or different.

In another subset of the above embodiments, the oligonucleotide or siRNAis double stranded; and the tetraGalNAc ligands are attached to theguide strand or the passenger strand of the oligonucleotide or siRNA atdifferent 2′-positions of the ribose rings.

In another subset of the above embodiments, the oligonucleotide or siRNAis double stranded; and the tetraGalNAc ligands are attached to theguide strand or the passenger strand of the oligonucleotide or siRNA atdifferent terminal 3′ and/or 5′-positions.

In another subset of the above embodiments, the oligonucleotide or siRNAis double stranded; and two or more tetraGalNAc ligands are attached toboth the guide strand and the passenger strand of the oligonucleotide orsiRNA at different 2′-positions of the ribose rings and/or at differentterminal 3′ and/or 5′-positions.

In another subset of the above embodiments, each linker is independentlyselected from Table 1.

In another subset of the above embodiments, each linker is independentlyselected from Table 2.

In another subset of the above embodiments, the oligonucleotide or siRNAis double stranded; and the optional targeting ligands, solubilizingagents, pharmacokinetics enhancing agents, lipids, and/or masking agentsare attached to the same or different strands of the oligonucleotide orsiRNA.

In one embodiment, a modular composition comprises 1) a double strandedsiRNA; 2) 1-8 tetraGalNAc ligands of Formula (IV), (V) or (VI):

3) 1-16 linkers independently selected from Table 1, which may be thesame or different; and, optionally, 4) 1-8 targeting ligands,solubilizing agents, pharmacokinetics enhancing agents, lipids, and/ormasking agents.

In another embodiment, a modular composition comprises 1) a doublestranded siRNA; 2) 1-4 tetraGalNAc ligands of Formula (IV), (V) or (VI);3) 1-8 linkers independently selected from Table 1, which may be thesame or different; and, optionally, 4) 1-4 targeting ligands,solubilizing agents, pharmacokinetics enhancing agents, lipids, and/ormasking agents; wherein the tetraGalNAc ligands are attached to thesiRNA at different 2′-positions of the ribose rings and/or at differentterminal 3′ and/or 5′-positions of the siRNA; and wherein thetetraGalNAc ligands are attached to the siRNA optionally via linkers.

In one subset of the above embodiments, the tetraGalNAc ligands areattached to the same strand of the siRNA via linkers.

In one embodiment, a modular composition comprises 1) a double strandedsiRNA; 2) 1-4 tetraGalNAc ligands of Formula (V); 3) 1-8 linkersindependently selected from Table 2, which may be the same or different;and, optionally, 4) 1-4 targeting ligands, solubilizing agents,pharmacokinetics enhancing agents, lipids, and/or masking agents;wherein the tetraGalNAc ligands are attached to the siRNA at different2′-positions of the ribose rings and/or at different terminal 3′ and/or5′-positions of the siRNA; and wherein the tetraGalNAc ligands areattached to the siRNA via linkers.

In one subset of the above embodiment, the tetraGalNAc ligands areattached to the same strand of the siRNA via linkers.

DETAILED DESCRIPTION OF THE INVENTION

Disclosed herein are single chemical conjugates comprising a singlestranded or double stranded oligonucleotide; and one or more tetraGalNAcligands of Formula (I), which may be the same or different:

wherein X is —O—, —S—, —CR¹R²— or —NR¹—, wherein R¹ and R² are eachindependently selected from the group consisting of hydrogen andC1-C6alkyl; n is 1, 2, 3, or 4; and the bond with “

” indicates the point of attachment. Other functionalities, such astargeting ligands, solubilizing agents, pharmacokinetics enhancingagents, lipids, and/or masking agents are optionally present. In oneembodiment, R¹ and R² are each independently selected from the groupconsisting of hydrogen, methyl and ethyl. In another embodiment, R¹ andR² are each hydrogen.

In one embodiment, the tetraGalNAc ligand has Formula (II) wherein X,R¹, R² and n are as defined above. In another embodiment, thetetraGalNAc ligand has Formula (III) wherein X, R¹, R² and n are asdefined above:

In one embodiment, the oligonucleotide is a short interfering RNA(siRNA). In another embodiment, the siRNA is a single stranded siRNA. Inanother embodiment, the siRNA is a double stranded siRNA.

The use of the tetraGalNAc disclosed herein may provide effectivedelivery of the oligonuleotide or siRNA by directing the modularcomposition to a particular cell. For example, the targeting ligand mayspecifically or non-specifically bind with a molecule on the surface ofa target cell and facilitate uptake of the ligand-siRNA conjugate.

A linker may be present between each tetraGalNAc and theoligonucleotide. The linkers are attached to the oligonucleotide atdifferent 2′-positions of the ribose rings and/or the terminal 3′ and/or5′-positions of the oligonucleotide.

In one embodiment, a modular composition comprises 1) a single strandedor double stranded oligonucleotide; 2) one or more tetraGalNAc ligandsof Formula (I), which may be the same or different, wherein X is —O—,—S—, —H₂— or —NH—; n is 1, 2, 3, or 4; and the bond with “

” indicates the point of attachment; optionally, 3) one or more linkers,which may be the same or different; and optionally, 4) one or moretargeting ligands, solubilizing agents, pharmacokinetics enhancingagents, lipids, and/or masking agents.

In another embodiment, a modular composition comprises 1) a singlestranded or double stranded oligonucleotide; 2) 1-8 tetraGalNAc ligandsof Formula (I), (II) or (III), which may be the same or different,wherein X is —O—, —S—, —H₂— or —NH—; n is 1, 2, 3, or 4; 3) 1-16linkers, which may be the same or different; and optionally, 4) 1-8targeting ligands, solubilizing agents, pharmacokinetics enhancingagents, lipids, and/or masking agents.

In another embodiment, a modular composition comprises 1) a singlestranded or double stranded siRNA; 2) 1-8 tetraGalNAc ligands of Formula(I), (II) or (III), which may be the same or different, wherein X is—O—, —S—, —H₂— or —NH—; n is 1, 2, 3, or 4; 3) 1-16 linkers, which maybe the same or different; and optionally, 4) 1-8 targeting ligands,solubilizing agents, pharmacokinetics enhancing agents, lipids, and/ormasking agents.

In one subset of the above embodiments, the tetraGalNAc ligands areattached to the oligonucleotide or siRNA at different 2′-positions ofthe ribose rings and/or at different terminal 3′ and/or 5′-positions ofthe oligonucleotide or siRNA. In another subset of the aboveembodiments, the tetraGalNAc ligands are attached to the oligonucleotideor siRNA optionally via linkers. In one embodiment, the linkers arepresent.

In another subset of the above embodiments, the tetraGalNAc ligands areattached to the oligonucleotide or siRNA at different 2′-positions ofthe ribose rings and/or at different terminal 3′ and/or 5′-positions ofthe oligonucleotide or siRNA; and the tetraGalNAc ligands are attachedto the oligonucleotide or siRNA via linkers.

In another subset of the above embodiments, the tetraGalNAc ligands areattached to the oligonucleotide or siRNA via linkers and the linkers areattached to the oligonucleotide or siRNA at different 2′-positions ofthe ribose rings.

In another subset of the above embodiments, the tetraGalNAc ligands areattached to the oligonucleotide or siRNA via linkers and the linkers areattached to the oligonucleotide or siRNA at different terminal 3′ and/or5′-positions of the oligonucleotide.

In another subset of the above embodiments, X is —O—, —S—, or —H₂—. Inanother embodiment, X is —O— or —CH₂—. In another embodiment, n is 1, 2or 3. In another embodiment, X is —O— and n is 1 or 2. In anotherembodiment, X is —CH₂— and n is 1 or 2. In another embodiment, X is —O—and n is 1. In yet another embodiment, X is —CH₂— and n is 1.

In another subset of the above embodiments, the oligonucleotide or siRNAis single stranded. In another embodiment, the oligonucleotide or siRNAis double stranded. In another subset of the above embodiments, thecomposition comprises 1-6 tetraGalNAc ligands. In another embodiment,the composition comprises 1-4 tetraGalNAc ligands. In anotherembodiment, the composition comprises 1-2 tetraGalNAc ligands. In yetanother embodiment, the composition comprises 1 tetraGalNAc ligand.

In another subset of the above embodiments, the oligonucleotide or siRNAis double stranded and the tetraGalNAc ligands are attached to the guidestrand at different 2′-positions of the ribose rings.

In another subset of the above embodiments, the oligonucleotide or siRNAis double stranded the tetraGalNAc ligands are attached to the guidestrand at different terminal 3′ and/or 5′-positions.

In another subset of the above embodiments, the oligonucleotide or siRNAis double stranded and the tetraGalNAc ligands are attached to thepassenger strand at different 2′-positions of the ribose rings.

In another subset of the above embodiments, the oligonucleotide or siRNAis double stranded and the tetraGalNAc ligands are attached to thepassenger strand at different terminal 3′ and/or 5′-positions.

In another subset of the above embodiments, the oligonucleotide or siRNAis double stranded and the tetraGalNAc ligands are attached to both theguide strand and the passenger strand at different 2′-positions of theribose rings and/or different terminal 3′ and/or 5′-positions.

In another subset of the above embodiments, the oligonucleotide or siRNAis double stranded and the tetraGalNAc ligands are attached to the samestrand.

In another subset of the above embodiments, the oligonucleotide or siRNAis double stranded and the tetraGalNAc ligands are attached to differentstrands.

In another subset of the above embodiments, the oligonucleotide or siRNAis double stranded and the optional targeting ligands, solubilizingagents, pharmacokinetics enhancing agents, lipids, and/or masking agentsare attached to the same or different strands.

In another subset of the above embodiments, the oligonucleotide or siRNAis double stranded and the optional targeting ligands, solubilizingagents, pharmacokinetics enhancing agents, lipids, and/or masking agentsare attached to the same or different strands via linkers. In oneembodiment, each linker is independently selected Table 1. In anotherembodiment, each linker is independently selected Table 2.

In one embodiment, a modular composition comprises 1) a single strandedor double stranded siRNA; 2) 1-8 tetraGalNAc ligands of Formula (I),(II) or (III), which may be the same or different; wherein X is —O—,—S—, —H₂— or —NH—; and n is 1, 2, 3, or 4; 3) 1-16 linkers, which may bethe same or different; and optionally, 4) 1-8 targeting ligands,solubilizing agents, pharmacokinetics enhancing agents, lipids, and/ormasking agents; wherein the tetraGalNAc ligands are attached to thesiRNA at different 2′-positions of the ribose rings and/or at differentterminal 3′ and/or 5′-positions of the siRNA; and wherein thetetraGalNAc ligands are attached to the siRNA optionally via linkers. Inone embodiment, the linkers are present. In another embodiment, X is—O—, —S—, or —H₂—, and n is 1, 2 or 3. In another embodiment, X is —O—or —H₂—, and n is 1 or 2.

In another embodiment, a modular composition comprises 1) a doublestranded siRNA; 2) 1-6 tetraGalNAc ligands of Formula (I), which may bethe same or different; wherein X is —O—, —S—, or —H₂-; and n is 1, 2 or3; 3) 1-18 linkers, which may be the same or different; and optionally,4) 1-6 targeting ligands, solubilizing agents, pharmacokineticsenhancing agents, lipids, and/or masking agents; wherein the tetraGalNAcligands are attached to the siRNA at different 2′-positions of theribose rings and/or at different terminal 3′ and/or 5′-positions of thesiRNA; and wherein the tetraGalNAc ligands are attached to the siRNAoptionally via linkers. In one embodiment, the linkers are present. Inanother embodiment, X is —O—, —S—, or —H₂— and n is 1 or 2. In anotherembodiment, the linkers are independently selected from Table 1. Inanother embodiment, the linkers are independently selected from Table 2.

In another embodiment, a modular composition comprises 1) a doublestranded siRNA; 2) 1-4 tetraGalNAc ligands of Formula (I), which may bethe same or different; wherein X is —O—, —S—, or —H₂—; and n is 1 or 2;3) 1-8 linkers, which may be the same or different; and optionally, 4)1-4 targeting ligands, solubilizing agents, pharmacokinetics enhancingagents, lipids, and/or masking agents; wherein the tetraGalNAc ligandsare attached to the siRNA at different 2′-positions of the ribose ringsand/or at different terminal 3′ and/or 5′-positions of the siRNA; andwherein the tetraGalNAc ligands are attached to the siRNA via linkers.In one embodiment, X is —O— or —H₂— and n is 1 or 2. In anotherembodiment, the linkers are independently selected from Table 1. Inanother embodiment, the linkers are independently selected from Table 2.

In another embodiment, a modular composition comprises 1) a doublestranded siRNA; 2) 1-4 tetraGalNAc ligands of Formula (IV), (V) or (VI):

3) 1-8 linkers independently selected from Table 1, which may be thesame or different; and optionally, 4) 1-4 targeting ligands,solubilizing agents, pharmacokinetics enhancing agents, lipids, and/ormasking agents; wherein the tetraGalNAc ligands are attached to thesiRNA at different 2′-positions of the ribose rings and/or at differentterminal 3′ and/or 5′-positions of the siRNA; and wherein thetetraGalNAc ligands are attached to the siRNA via linkers.

In another embodiment, a modular composition comprises 1) a doublestranded siRNA; 2) 1-4 tetraGalNAc ligands of Formula (V); 3) 1-8linkers independently selected from Table 2, which may be the same ordifferent; and optionally, 4) 1-4 targeting ligands, solubilizingagents, pharmacokinetics enhancing agents, lipids, and/or maskingagents; wherein the tetraGalNAc ligands are attached to the siRNA atdifferent 2′-positions of the ribose rings and/or at different terminal3′ and/or 5′-positions of the siRNA; and wherein the tetraGalNAc ligandsare attached to the siRNA via linkers.

In one subset of the above embodiments, the tetraGalNAc ligands areattached to the siRNA via linkers; and wherein the tetraGalNAc ligandsare attached to the same strand.

In another subset of the above embodiments, the tetraGalNAc ligands areattached to the siRNA via linkers; and wherein the tetraGalNAc ligandsare attached to different strands.

To illustrate the invention via cartoon, the invention features amodular composition, comprising an oligonucleotide ([O₁][O₂][O₃] . . .[O_(n)]), one or more linkers (L), one or more tetraGalNAc ligands (G),and one or more optional lipid(s) (X), targeting ligand(s) (X), and/orsolubilizing group(s) (X).

In an embodiment, the modular composition may have the formula:

G-L-[O₁][O₂][O₃] . . . [O_(n)].

In another embodiment, the modular composition may have the formula:

G-L-[O₁][O₂][O₃] . . . [O_(n)]—X.

Non-limiting examples of modular compositions comprising double strandedoligonucleotides with terminal conjugations are:

Non-limiting examples of modular compositions comprising double strandedoligonucleotides with internal conjugations are:

These examples are used as illustration only. One skilled in the artwill recognize that a variety of permutations for placing the desiredcomponents on the passenger and guide strands exist.

Any number of linkers, and therefore any number of tetraGalNAc ligands,can be attached to the oligonucleotide. A preferred range of numbers oflinkers is from 1-16. A more preferred range of numbers of linkers isfrom 1-8, or more specifically, 1-4. A preferred range of numbers oftetraGalNAc ligands is from 1-8. A more preferred range of numbers ofpeptides is from 1-8, or more specifically, 1-4.

The two strands contain n and n′ nucleotides respectively. The numbers nand n′ can be equal or different. The numbers are integers ranging from8 to 50. Preferably, the numbers are integers ranging from 12-28. Morepreferably, the numbers are integers ranging from 19-21.

As an example, each nucleotide [O_(n)] or [O_(n′)], that contains alinker (L-G and/or L-X) has generic structures shown in the followingcartoon:

For each nucleotide, 1) E=oxygen (O) or sulfur (S); 2) Base=A, U, G orC, which can be modified or unmodified; 3) D is the connection pointbetween ribose ring and linker L, D=oxygen (O), sulfur (S, S(O) orS(O)₂), nitrogen (N—R, wherein R═H, alkyl, L-P or L-X), carbon (CH—R,wherein R═H, alkyl, L-P, or L-X), or phosphorus (P(O)R or P(O)(OR),wherein R=alkyl, L-G, or L-X). Preferably, D=oxygen (O).

The two nucleotides [O_(n-1)] and [O_(n)] or [O_(n′-1)] and [O_(n′)] areconnected via phosphodiester or thio-phosphodiester bonds.

When the oligonucleotide is a double-stranded oligonucleotide, the “G-L”and the lipid, targeting ligand, and/or solubilizing group may belocated on the same strand or on different strands.

In some embodiments, the “G-L” and the lipid, targeting ligand, and/orsolubilizing group are on the same strand.

In some embodiments, the “G-L” and the lipid, targeting ligand, and/orsolubilizing group are on the passenger strand.

In some embodiments, the “G-L” and the lipid, targeting ligand, and/orsolubilizing group are on the guide strand.

In some embodiments, the “G-L” and the lipid, targeting ligand, and/orsolubilizing group are located on different strands.

In some embodiments, the “G-L” is on the passenger strand while thelipid, targeting ligand, and/or solubilizing group is on the guidestrand.

In some embodiments, the “G-L” and the lipid, targeting ligand, and/orsolubilizing group are on different strands but on the same terminal endof the double-stranded oligonucleotide.

In some embodiments, the “G-L” and the lipid, targeting ligand, and/orsolubilizing group are on different strands and on the opposite terminalends of the double-stranded oligonucleotide.

In some embodiments, an additional “G-L” of identical or differentnature can be used in place of the lipid, targeting ligand, and/orsolubilizing group noted in the above embodiments.

In some embodiments, the “G-L” can be located on multiple terminal endsof either the passenger or guide strand and the the lipid, targetingligand, and/or solubilizing group can be located on the remainingterminal ends of the passenger and guide strands.

In some embodiments, one “G-L” and two or more lipids, targetingligands, and/or solubilizing groups are present in the oligonucleotide.

In some embodiments, two or more “G-L” and two or more lipids, targetingligands and/or solubilizing groups are present in the oligonucleotide.

In some embodiments, when the oligonucleotide is a double-strandedoligonucleotide and multiple “G-L” components and/or lipids, targetingligands, and/or solubilizing groups are present, such multiple “G-L”components and/or lipids, targeting ligands, and/or solubilizing groupsmay all be present in one strand or both strands of the double strandedoligonucleotide.

When multiple “G-L” components and/or lipids, targeting ligands, and/orsolubilizing groups are present, they may all be the same or different.

In some embodiments, the “G-L” are on internal nucleotides only (i.e.excluding the 3′- and 5′-terminal ends of the oligonucleotide).

In another aspect, the invention includes a method of delivering anoligonucleotide or siRNA to a cell. The method includes (a) providing orobtaining a modular composition disclosed herein; (b) contacting a cellwith the modular composition; and (c) allowing the cell to internalizethe modular composition.

The method can be performed in vitro, ex vivo or in vivo, e.g., to treata subject identified as being in need of an oligonucleotide or siRNA. Asubject in need of said oligonucleotide is a subject, e.g., a human, inneed of having the expression of a gene or genes, e.g., a gene relatedto a disorder, downregulated or silenced.

In one aspect, the invention provides a method for inhibiting theexpression of one or more genes. The method comprising contacting one ormore cells with an effective amount of an oligonucleotide of theinvention, wherein the effective amount is an amount that suppresses theexpression of the one or more genes. The method can be performed invitro, ex vivo or in vivo.

The methods and compositions of the invention, e.g., the modularcomposition described herein, can be used with any oligonucleotides orsiRNAs known in the art. In addition, the methods and compositions ofthe invention can be used for the treatment of any disease or disorderknown in the art, and for the treatment of any subject, e.g., anyanimal, any mammal, such as any human. One of ordinary skill in the artwill also recognize that the methods and compositions of the inventionmay be used for the treatment of any disease that would benefit fromdownregulating or silencing a gene or genes.

The methods and compositions of the invention, e.g., the modularcomposition described herein, may be used with any dosage and/orformulation described herein, or any dosage or formulation known in theart. In addition to the routes of administration described herein, aperson skilled in the art will also appreciate that other routes ofadministration may be used to administer the modular composition of theinvention.

Oligonucleotide

An “oligonucleotide” as used herein, is a double stranded or singlestranded, unmodified or modified RNA or DNA Examples of modified RNAsinclude those which have greater resistance to nuclease degradation thando unmodified RNAs. Further examples include those which have a 2′ sugarmodification, a base modification, a modification in a single strandoverhang, for example a 3′ single strand overhang, or, particularly ifsingle stranded, a 5′ modification which includes one or more phosphategroups or one or more analogs of a phosphate group. Examples and afurther description of oligonucleotides can be found in WO2009/126933,which is hereby incorporated by reference.

In an embodiment, an oligonucleotide is an antisense, miRNA, peptidenucleic acid (PNA), poly-morpholino (PMO) or siRNA. The preferredoligonucleotide is an siRNA. Another preferred oligonuleotide is thepassenger strand of an siRNA. Another preferred oligonucleotide is theguide strand of an siRNA.

siRNA

siRNA directs the sequence-specific silencing of mRNA through a processknown as RNA interference (RNAi). The process occurs in a wide varietyof organisms, including mammals and other vertebrates. Methods forpreparing and administering siRNA and their use for specificallyinactivating gene function are known. siRNA includes modified andunmodified siRNA. Examples and a further description of siRNA can befound in WO2009/126933, which is hereby incorporated by reference.

A number of exemplary routes of delivery are known that can be used toadminister siRNA to a subject. In addition, the siRNA can be formulatedaccording to any exemplary method known in the art. Examples and afurther description of siRNA formulation and administration can be foundin WO2009/126933, which is hereby incorporated by reference.

The phrases “short interfering nucleic acid”, “siNA”, “short interferingRNA”, “siRNA”, “short interfering nucleic acid molecule”,“oligonucleotide”, “short interfering oligonucleotide molecule”, or“chemically modified short interfering nucleic acid molecule” refer toany nucleic acid molecule capable of inhibiting or down regulating geneexpression or viral replication by mediating RNA interference (“RNAi”)or gene silencing in a sequence-specific manner. These terms can referto both individual nucleic acid molecules, a plurality of such nucleicacid molecules, or pools of such nucleic acid molecules. The siNA can bea double-stranded nucleic acid molecule comprising self-complementarysense and antisense strands, wherein the antisense strand comprises anucleotide sequence that is complementary to a nucleotide sequence in atarget nucleic acid molecule or a portion thereof and the sense strandcomprises a nucleotide sequence corresponding to the target nucleic acidsequence or a portion thereof. The siNA can be a polynucleotide with aduplex, asymmetric duplex, hairpin or asymmetric hairpin secondarystructure, having self-complementary sense and antisense regions,wherein the antisense region comprises a nucleotide sequence that iscomplementary to a nucleotide sequence in a separate target nucleic acidmolecule or a portion thereof and the sense region comprises anucleotide sequence corresponding to the target nucleic acid sequence ora portion thereof. The siNA can be a circular single-strandedpolynucleotide having two or more loop structures and a stem comprisingself-complementary sense and antisense regions, wherein the antisenseregion comprises nucleotide sequence that is complementary to anucleotide sequence in a target nucleic acid molecule or a portionthereof and the sense region comprises a nucleotide sequencecorresponding to the target nucleic acid sequence or a portion thereof,and wherein the circular polynucleotide can be processed either in vivoor in vitro to generate an active siNA molecule capable of mediatingRNAi. The siNA can also comprise a single-stranded polynucleotide havinga nucleotide sequence complementary to nucleotide sequence in a targetnucleic acid molecule or a portion thereof (for example, where such siNAmolecule does not require the presence within the siNA molecule of anucleotide sequence corresponding to the target nucleic acid sequence ora portion thereof), wherein the single-stranded polynucleotide canfurther comprise a terminal phosphate group, such as a 5′-phosphate (seefor example, Martinez et al., 2002, Cell, 110, 563-574 and Schwarz etal., 2002, Molecular Cell, 10, 537-568), or 5′,3′-diphosphate.

Linkers

The covalent linkages between the tetraGalNAc and the oligonucleotide orsiRNA of the modular composition may be mediated by a linker. Thislinker may be cleavable or non-cleavable, depending on the application.In certain embodiments, a cleavable linker may be used to release theoligonucleotide after transport from the endosome to the cytoplasm. Theintended nature of the conjugation or coupling interaction, or thedesired biological effect, will determine the choice of linker group.Linker groups may be combined or branched to provide more complexarchitectures. Suitable linkers include those as described inWO2009/126933, which is hereby incorporated by reference.

In one embodiment, the linkers of the instant invention are shown inTable 1:

TABLE 1

R = H, Boc, Cbz, Ac, PEG, lipid, targeting ligand, linker(s) and/orpeptide(s). n = 0 to 750. “nucleotide” can be substituted withnon-nucleotide moiety such as abasic or linkers as are generally knownin the art.

In another embodiment, the preferred linkers are shown in Table 2:

TABLE 2

R = H, Boc, Cbz, Ac, PEG, lipid, targeting ligand, linker(s) and/orpeptide(s). n = 0 to 750. “nucleotide” can be substituted withnon-nucleotide moiety such as abasic or linkers as are generally knownin the art.

Commercial linkers are available from various suppliers such as Pierceor Quanta Biodesign including combinations of said linkers. In addition,commercial linkers attached via phosphate bonds can be usedindependently as linkers or in combination with said linker. The linkersmay also be combined to produce more complex branched architecturesaccommodating from 1 to 8 tetraGalNAc ligands as illustrated in one suchexample below:

Targeting Ligands

The modular compositions of the present invention may comprise atargeting ligand. In some embodiments, this targeting ligand may directthe modular composition to a particular cell. For example, the targetingligand may specifically or non-specifically bind with a molecule on thesurface of a target cell. The targeting moiety can be a molecule with aspecific affinity for a target cell. Targeting moieties can includeantibodies directed against a protein found on the surface of a targetcell, or the ligand or a receptor-binding portion of a ligand for amolecule found on the surface of a target cell. Examples and a furtherdescription of targeting ligands can be found in WO2009/126933, which ishereby incorporated by reference.

The targeting ligands are selected from the group consisting of anantibody, a ligand-binding portion of a receptor, a ligand for areceptor, an aptamer, D-galactose, N-acetyl-D-galactose (GalNAc),multivalent N-acetyl-D-galactose, D-mannose, cholesterol, a fatty acid,a lipoprotein, folate, thyrotropin, melanotropin, surfactant protein A,mucin, carbohydrate, multivalent lactose, multivalent galactose,N-acetyl-galactosamine, N-acetyl-glucosamine, multivalent mannose,multivalent fructose, glycosylated polyaminoacids, transferin,bisphosphonate, polyglutamate, polyaspartate, a lipophilic moiety thatenhances plasma protein binding, a steroid, bile acid, vitamin B12,biotin, an RGD peptide, an RGD peptide mimic, ibuprofen, naproxen,aspirin, folate, and analogs and derivatives thereof.

The preferred targeting ligands are selected from the group consistingof D-galactose, N-acetyl-D-galactose (GalNAc), GalNAc2, and GalNAc3,cholesterol, folate, and analogs and derivatives thereof.

Lipids

Lipophilic moieties, such as cholesterol or fatty acids, when attachedto highly hydrophilic molecules such as nucleic acids can substantiallyenhance plasma protein binding and consequently circulation half life.In addition, lipophilic groups can increase cellular uptake. Forexample, lipids can bind to certain plasma proteins, such aslipoproteins, which have consequently been shown to increase uptake inspecific tissues expressing the corresponding lipoprotein receptors(e.g., LDL-receptor or the scavenger receptor SR-B1). Lipophilicconjugates can also be considered as a targeted delivery approach andtheir intracellular trafficking could potentially be further improved bythe combination with endosomolytic agents.

Exemplary lipophilic moieties that enhance plasma protein bindinginclude, but are not limited to, sterols, cholesterol, fatty acids,cholic acid, lithocholic acid, dialkylglycerides, diacylglyceride,phospholipids, sphingolipids, adamantane acetic acid, 1-pyrene butyricacid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexylgroup, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecylgroup, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid,O3-(oleoyl)cholenic acid, dimethoxytrityl, phenoxazine, aspirin,naproxen, ibuprofen, vitamin E and biotin etc. Examples and a furtherdescription of lipids can be found in WO2009/126933, which is herebyincorporated by reference.

The preferred lipid is cholesterol.

Solubilizing Agents

The modular composition may comprise one or more other moieties/ligandsthat may enhance aqueous solubility, circulation half life and/orcellular uptake. These can include naturally occurring substances, suchas a protein (e.g., human serum albumin (HSA), low-density lipoprotein(LDL), high-density lipoprotein (HDL), or globulin); or a carbohydrate(e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin orhyaluronic acid). These moieties may also be a recombinant or syntheticmolecule, such as a synthetic polymer or synthetic polyamino acids.Examples include polylysine (PLL), poly L-aspartic acid, poly L-glutamicacid, styrene-maleic acid anhydride copolymer,poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydridecopolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA),polyethylene glycol (PEG, e.g., PEG-0.5K, PEG-2K, PEG-5K, PEG-10K,PEG-12K, PEG-15K, PEG-20K, PEG-40K), methyl-PEG (mPEG), [mPEG]2,polyvinyl alcohol (PVA), polyurethane, poly(2 ethylacrylic acid),N-isopropylacrylamide polymers, or polyphosphazine. Examples and afurther description of solubilizing agents can be found inWO2009/126933, which is hereby incorporated by reference.

The preferred solubilizing group is PEG 0.5K to 30K.

Method of Treatment

In one aspect, the invention features, a method of treating a subject atrisk for or afflicted with a disease that may benefit from theadministration of the modular composition of the invention. The methodcomprises administering the modular composition of the invention to asubject in need thereof, thereby treating the subject. Theoligonucleotide that is administered will depend on the disease beingtreated. See WO2009/126933 for additional details regarding methods oftreatments for specific indications.

Formulation

There are numerous methods for preparing conjugates of oligonucleotidecompounds. The techniques should be familiar to those skilled in theart. A useful reference for such reactions is Bioconjugate Techniques,Hermanson, G. T., Academic Press, San Diego, Calif., 1996. Otherreferences include WO2005/041859; WO2008/036825 and WO2009/126933.

EXAMPLES

The invention is further illustrated by the following examples, whichshould not be construed as further limiting. The contents of allreferences, pending patent applications and published patents, citedthroughout this application are hereby expressly incorporated byreference. The siRNAs described herein were designed to target theubiquitously expressed gene SSB (Sjogren syndrome antigen B;NM_(—)009278.4).

Linker groups may be connected to the oligonucleotide or siRNA strand(s)at a linkage attachment point (LAP) and may include anycarbon-containing moiety, in some embodiments having at least one oxygenatom, at least one phosphorous atom, and/or at least one nitrogen atom.In some embodiments, the phosphorous atom forms part of a terminalphosphate, or phosphorothioate, group on the linker group, which mayserve as a connection point for the oligonucleotide strand. In certainembodiments, the nitrogen atom forms part of a terminal ether, ester,amino or amido (NHC(O)—) group on the linker group, which may serve as aconnection point for the linkers of interest, endosomolytic unit, cellpenetrating peptide, solubilizing group, lipid, targeting group, oradditional linkers of interest. These terminal linker groups include,but are not limited to, a C₆ hexyl, C₅ secondary-hydroxy, C₃ thiol or C₆thiol moiety. An example from the RNA sequences described below is C6hexyl: [(CH₂)₆ NH₂].

Preparations of tetraGalNAc ligands and tetraGalNAc-siRNA conjugates aredescribed below in the Examples and synthetic schemes. Specific siRNAsequences used for the compounds and/or conjugates described below areshown in Table 3.

Gene Duplex Entry Target Strand Sequence Code 1 ApoB Passenger[6amiL][iB][omeC][omeU] 51 [omeU][omeU][fluA][fluA][omeC][fluA][fluA][omeU] [omeU][omeC][omeC][omeU] [fluG][fluA][fluA][fluA][omeU][dTs] [dT][iB] ApoB Guide [rAs][rUs][rUs][omeU][omeC][fluA][fluG][fluG][fluA][fluA] [omeU][omeU][fluG][fluU][omeU][fluA][fluA][fluG] [omeUs][fluA][omeU] 2 ApoB Passenger[6amiL][iB][omeC][omeU] 52 [omeU][omeU] [fluA][fluA][omeC][fluA][fluA][omeU][omeU][omeC] [omeC][omeU][fluG] [fluA][fluA][fluA][omeU][dTs]dT[iB][6amiL] ApoB Guide [rAs][rUs][rUs][omeU][omeC][fluA][fluG][fluG][fluA][fluA] [omeU][omeU][fluG][fluU][omeU][fluA][fluA][fluG] [omeUs][fluA][omeU] 3 ApoB Passenger[6amiL][iB][omeC][omeU] 53 [clickU][omeU][fluA][fluA][omeC][fluA][fluA][omeU] [omeU][omeC][omeC][omeU][fluG][fluA][clickA][fluA] [omeU][dTs]dT[iB] ApoB Guide[rAs][rUs][rUs][omeU] [omeC][fluA] [fluG][fluG][fluA][fluA][omeU][omeU][fluG][fluU][omeU] [fluA][fluA][fluA][fluG] [omeUs][omeU] 4ApoB Passenger [iB][omeC][omeU] 54 [omeU][omeU][fluA][fluA][omeC][fluA][flu A][omeU][omeU][omeC][omeC][omeU][fluG][fluA][fluA][fluA] [omeU][dTs]dT[iB][6amiL] ApoB Guide[rAs][rUs][rUs][omeU][omeC] [fluA][fluG][fluG][fluA][fluA] [omeU][omeU][fluG][fluU][omeU] [fluA][fluA][fluA][fluG] [omeUs][omeU] 5 ApoBPassenger [6amiL][iB][omeC][omeU][ome 55 U][omeU][fluA][fluA][omeC][fluA][fluA][omeU][omeU][omeC] [omeC][omeU][fluG][fluA][fluA][fluA][omeU][dTs]dT[iB] ApoB Guide [rAs][rUs][rUs][omeU][omeC][fluA][fluG][fluG][fluA][fluA][om eU][omeU][fluG][fluU][propargylU][fluA] [fluA][fluA][fluG][om eUs][omeU] 6 SSB Passenger[6amiL][iB][fluA][omeC][fluA] 56 [fluA][omeC][fluA][fluG][fluA][omeC][omeU][omeU] [omeU][fluA] [fluA][omeU][fluG][omeU][fluA][fluA][dTs]dT[iB] SSB Guide [rUs][rUs][rAs][omeC] [fluA][omeU][omeU][fluA][fluA][fluA][fl uG][omeU][omeC][fluU][fluG][omeU][omeU][fluG][omeU] [omeUs][omeU] 7 CTNNB1 Passenger[6amiL][iB][omeC] 57 [omeU][clickG] [omeU][omeU][fluG][fluG][fluA][omeU][omeU][fluG][fluA] [omeU][omeU][omeC][fluG][clickA][fluA][fluA] [omeUs][omeU][iB] CTNNB1 Guide[omeUs][fluUs][omeUs][fluC] [omeG][fluA][omeA][fluU][omeC][fluA][omeA][fluU][omeC] [fluC][omeA][fluA][omeC][fluA][omeG][omeUs][omeU] 8 CTNNB1 Passenger [6amiL][iB][omeC][omeU] 58[clickG][omeU][omeU][fluG] [fluG][fluA][omeU][omeU][fluG][fluA][omeU][omeU] [omeC][fluG][clickA][fluA][fluA] [omeUs][omeU][iB]CTNNB1 Guide [omeUs][fluUs][omeUs][fluC][ome G][fluA][omeA][fluU][omeC][fluA][omeA][fluU][omeC] [fluC][clickA][fluA][omeC][fluA][omeG][omeUs][omeU] As used herein, ome = 2' methoxy; flu =2' fluoro;click = 2' propagyl; iB =inverted abasic; “s” subscript=phosphorothioate; and r =2' ribo; 6amil = n-hexylamino.

In Vitro and in Vivo Activity Data

Using the method as described later, the in vivo and in vitro data arepresented in Table 4.

TABLE 4 In vitro and In Vivo Activity Starting siRNA Dose (mpk) Route ofIn vivo % IC50 w/LF2K in ASGR binding Entry Compound sequence codeAdministration KD (72 h) HEK-Luc [pM] IC50 nM 1   10a-1 51 5, 15 SC33.6; 69.5 15.44 36.7 2   10b-1 54 SC 5, 15; IV 15 42, 49, 13 19.64 18.13   10-2 56 5, 50 SC 40, 56 (24 h) 23.4 4   10-3 57 1, 2.5, 5 SC 20, 45,60 52 (HepG2) 5   17a-1 51 5 SC; 15 IV 11, 5  20.16 49.1 6   17b-1 54 5SC; 15 IV 12, 22 43.96 33.3 7   19-1 52 5; 15 SC 32; 68 24.04 3.6 8 2953 15 SC; 15 IV  43, 0  17.83 22 9 36 58 1, 2.5, 5 SC 16, 43, 56 10 3758 1, 2.5, 5 SC 16, 32, 40 11 38 51 5 SC, 15 IV 36, 33 71 17 12 39 51 5SC, 15 IV 19, 31 46.8 44 13 40 51 5, 15 SC 33, 62 76.8 77 14 41 51 5, 15SC 28, 74 98.6 134 15 42 51 5, 15 SC 19, 73 309.7 135 16 43 51 5, 15 SC 8, 73 64.8 45 17 44 51 5, 15 SC 31, 73 67.1 66 18 45 51 5 SC, 15 IV 20,4  73.4 11 19   48a-1 51 5, 15 SC 10.24; 59.93 23.43 20   48b-1 53 5, 15SC 19.87; 42.08 57.96 21 51 55 5; 15   40; 45 1838.47 94.8

Note that the siRNA depictions below are for illustrative purposes.Specific sequence information can be found in Table 3.

Examples 1-2 Synthesis of TetraGalNAc Ligand Compounds 9 and 10

The following Scheme 1 was used to prepare TetraGalNAc Compounds 9 and10.

Synthesis of (2S)-2,6-bis[bis (prop-2-yn-1-yl)amino]hexanoic acid(Compound 1)

Into a 2000-mL 3-necked round-bottom flask purged and maintained with aninert atmosphere of nitrogen was placed a solution of(2S)-2,6-diaminohexanoic acid (50 g, 342.03 mmol, 1.00 equiv) inacetonitrile (1000 mL) and heated to 50° C. To this was added potassiumhydroxide (22.6 g, 0.4025 mol, 1.00 equiv, 85%). The resulting solutionwas stirred for 30 min. Then 3-bromoprop-1-yne (29.5 mL, 1.00 equiv) wasadded. The resulting solution was stirred for 1 hour at 50° C.additional potassium hydroxide (22.6 g, 0.4025 mol, 1.00 equiv) wasadded to the solution and stirred for 30 min at 50° C. To this was added3-bromoprop-1-yne (29.5 mL, 1.00 equiv). The resulting solution wasstirred for 1 hour. To this was added potassium hydroxide (22.6 g,0.4025 mol, 1.00 equiv) again. The resulting solution was stirred for 30min at 50° C., followed by addition of more 3-bromoprop-1-yne (29.5 mL,1.00 equiv). The resulting solution was stirred for 1 hour. To this wasadded potassium hydroxide (22.6 g, 0.4025 mol, 1.00 equiv). Theresulting solution was stirred for 30 min. To this was added3-bromoprop-1-yne (29.5 mL, 1.00 equiv). The resulting solution wasstirred for 3 hours. The reaction mixture was cooled to 25° C. with awater/ice bath. The solid was filtered out. The filtrate was adjusted topH 4 with HCl (6M). The solid was filtered out. The filtrate wasconcentrated under vacuum. The residue was applied onto a silica gelcolumn and eluted with dichloromethane/methanol (100:1-25:1). Thisresulted in (2S)-2, 6-bis[bis (prop-2-yn-1-yl)amino]hexanoic acid(Compound 1) as a light yellow oil. MS(ES, m/z): 297.2,[M-H]^(—1)HNMR(CDCl₃, 500 MHz, ppm): 3.62 (d, J=2.0 Hz, 4H), 3.52-3.49(m, 1H), 3.50 (d, J=2.4 Hz, 4H), 2.62 (t, J=7.1 Hz, 2H), 2.30 (t, J=2.4Hz, 2H), 2.27 (t, J=2.4 Hz, 2H),1.88-1.79 (m, 2H), 1.60-1.53 (m, 2H),1.52-1.43 (m, 2H).

Synthesis of 2-(2-hydroxyethoxy)ethyl 4-methylbenzenesulfonate (Compound3)

Into a 2000-mL 3-necked round-bottom flask purged and maintained with aninert atmosphere of nitrogen was placed a solution of2-(2-hydroxyethoxy)ethan-1-ol (Compound 2, 42.4 g, 399.55 mmol, 1.00equiv) in dichloromethane (1000 mL) and triethylamine (27.9 g, 275.72mmol, 0.25 equiv). To the above was added p-toluenesulfonyl chloride(19.1 g, 100.18 mmol, 0.50 equiv). After stirred for 1 h at 25° C., theresulting mixture was washed with 1×500 mL of aq. potassium hydrosulfate(1M) and 1×500 mL of aq. sodium bicarbonate (5%) respectively. Theorganic layer was dried over anhydrous sodium sulfate and concentratedunder vacuum. The residue was applied onto a silica gel column andeluted with dichloromethane/methanol (100:1). This resulted in2-(2-hydroxyethoxy)ethyl 4-methylbenzenesulfonate (Compound 3) as acolorless oil.

Synthesis of 2-(2-azidoethoxy)ethan-1-ol (Compound 4)

Into a 500-mL round-bottom flask purged and maintained with an inertatmosphere of nitrogen was placed a solution of2-(2-[[(4-2-(2-hydroxyethoxy)ethyl 4-methylbenzenesulfonate (Compound 3,50 g, 192.08 mmol, 1.00 equiv) in N,N-dimethylformamide (250 mL). Thiswas followed by the addition of sodium azide (18.79 g, 289.03 mmol, 1.50equiv) at 25° C. The resulting solution was stirred for 5 h at 100° C.in an oil bath. The reaction mixture was cooled and filtered. Thefiltrate was concentrated under vacuum. The residual solution wasdiluted with 1000 mL of dichloromethane and washed with 1×500 mL ofwater. The organic layer was dried over anhydrous sodium sulfate andconcentrated under vacuum. The residue was applied onto a silica gelcolumn and eluted with dichloromethane/methanol (80:1). This resulted in2-(2-azidoethoxy)ethan-1-ol (Compound 4) as a colorless oil.

¹HNMR (CDCl₃, 400 MHz, ppm): 3.42-3.45(t, J=4.8Hz, 2H), 3.63-3.65(t,J=4.8Hz, 2H), 3.71-3.74(t, J=4.8Hz, 2H), 3.71-3.79(m, 2H).Synthesis of(3R,4R,5R,6R)-3-acetamido-6-(acetoxymethyl)tetrahydro-2H-pyran-2,4,5-triyltriacetate (Compound 6)

Into a 2000-mL 3-necked round-bottom flask purged and maintained with aninert atmosphere of nitrogen was placed a solution of(3R,4R,5R,6R)-3-amino-6-(hydroxymethyl)tetrahydro-2H-pyran-2,4,5-triolhydrochloride (Compound 5, 120 g, 556.50 mmol, 1.00 equiv) in pyridine(1200 mL). This was followed by the addition of acetic anhydride (341.6g, 3.35 mol, 6.00 equiv) dropwise with stirring at 0° C. The resultingsolution was stirred overnight at 25° C. The reaction was then quenchedby the addition of 8000 mL of water/ice. The solid was collected byfiltration. This resulted in(3R,4R,5R,6R)-3-acetamido-6-(acetoxymethyl)tetrahydro-2H-pyran-2,4,5-triyltriacetate(Compound 6) as a white solid.

Synthesis of(3aR,5R,6R,7R,7aR)-5-(acetoxymethyl)-2-methyl-5,6,7,7a-tetrahydro-3aH-pyrano[3,2-d]oxazole-6,7-diyldiacetate (Compound 7)

Into a 2000-mL round-bottom flask purged and maintained with an inertatmosphere of nitrogen was placed a solution of(3R,4R,5R,6R)-3-acetamido-6-(acetoxymethyl)tetrahydro-2H-pyran-2,4,5-triyltriacetate (Compound 6, 30 g, 77.05 mmol, 1.00 equiv) in dichloromethane(1500 mL), then added iron (III) chloride (30 g, 184.95 mmol, 2.40equiv). The resulting mixture was stirred for 2 h at 25° C. The reactionwas then quenched by the addition of 1000 mL of water/ice. The organiclayer was washed with 1×1000 mL of sodium aq. bicarbonate and 1×1000 mLof water, dried over anhydrous sodium sulfate and concentrated undervacuum. This resulted in(3aR,5R,6R,7R,7aR)-5-(acetoxymethyl)-2-methyl-5,6,7,7a-tetrahydro-3aH-pyrano[3,2-d]oxazole-6,7-diyl diacetate (Compound 7) as yellow oil.

¹HNMR(CDCl₃, 300 MHz, ppm): 2.03(s, 9H), 2.12(s, 3H), 3.97-4.27(m, 4H),4.90-4.93(m, J =3.3Hz, 1H), 5.45-5.47(t, J=3.0Hz, 1H), 5.98-6.00(d,J=6.6Hz, 1H).Synthesis of(2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-[2-(2-azidoethoxy)ethoxy]tetrahydro-2H-pyran-3,4-diyldiacetate (Compound 8)

Into a 500-mL round-bottom flask purged and maintained with an inertatmosphere of nitrogen was placed a solution of(3aR,5R,6R,7R,7aR)-5-(acetoxymethyl)-2-methyl-5,6,7,7a-tetrahydro-3aH-pyrano[3,2-d]oxazole-6,7-diyldiacetate (Compound 7, 40 g, 121.47 mmol, 1.00 equiv) in1,2-dichloroethane (200 mL), 2-(2-azidoethoxy)ethan-1-ol (Compound 4,23.89 g, 182.18 mmol, 1.50 equiv). To the above several 4A zeolite wasadded. The resulting mixture was stirred for 1 h at 25° C. Thentrimethylsilyl trifluoromethanesulfonate (10.8 mL, 0.50 equiv) wasadded. After stirred overnight at 25° C., the reaction mixture wasdiluted with 500 mL of dichloromethane and washed with 1×500 mL ofwater, 1×500 ml, of aq. sodium bicarbonate and 1×500 mL of water. Theorganic layer was dried over anhydrous sodium sulfate and concentratedunder vacuum. The residue was applied onto a silica gel column andeluted with dichloromethane/methanol (100:1). This resulted in(2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-[2-(2-azidoethoxy)ethoxy]tetrahydro-2H-pyran-3,4-diyldiacetate (Compound 8) as a colorless oil.

MS(m/z): 461.1, [M+H]⁺

¹HNMR(CDCl₃, 500 MHz, ppm) 5.78 (d, J=8.90 Hz, 1H), 5.36 (d, J=2.9 Hz,1H), 5.22 (dd, J=11.2, 3.6 Hz, 1H), 4.77 (d, J=8.3 Hz, 1H), 4.19-4.12(m, 2H), 4.11-4.05 (m, 1H), 3.98-3.92 (m, 2H), 3.82-3.78 (m, 1H),3.71-3.63 (m, 4H), 3.49-3.38 (m, 2H), 2.16 (s, 3H), 2.05 (s, 3H), 2.01(s, 3H), 1.97 (s, 3H).Synthesis of(S)-2,6-bis(bis((1-(2-(2-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)methyl)amino)hexanoicacid (Compound 9, tetraGalNAc Acetate) (Ex. 1)

Into a 250-mL round bottom flask purged and maintained with an inertatmosphere of nitrogen was charged (2S)-2,6-bis[bis(prop-2-yn-1-yl)amino]hexanoic acid (Compound 1, 1.0 g, 1.0 equiv),(2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-[2-(2-azidoethoxy)ethoxy]tetrahydro-2H-pyran-3,4-diyldiacetate (Compound 8, 9.26 g, 6.0 equiv), anhydrous THF 50 mL,CuBr⁻SMe₂ (0.138 g, 0.20 equiv), and anhydrous DBU (1.5 ml, 3.0 equiv)in respective order. The resulting solution was stirred for 16 h at roomtemperature, quenched with acetic acid (0.75 mL, 4.0 equiv), treatedwith MP-TMT resin (Part No: 801472, from Biotage) (9 g), aged at roomtemperature for 16 h, filtered, and concentrated the filtrate to a foamsolid. The solid was then dissolved in CH₂Cl₂ (140 mL), and washed withAcOH/NaCl solution (140 mL). The AcOH/NaCl solution was prepared with 1mL AcOH and 100 mL 20% NaCl solution. The bottom organic layer wasconcentrated, and purified on a Si0₂ column (220 g), eluting withCH₂Cl₂/MeOH. This resulted in(S)-2,6-bis(bis((1-(2-(2-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethy)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)methyl)amino)hexanoicacid (Compound 9) as a white solid.

MS(m/z): 2139.5, [M+H]⁺

Synthesis of(S)-2,6-bis(bis((1-(2-(2-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)methyl)amino)hexanoicacid (Compound 10, TetraGalNAc) (Ex. 2)

Into a 250-mL round-bottom flask purged and maintained with an inertatmosphere of nitrogen was charged(S)-2,6-bis(bis((1-(2-(2-4(2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)methyl)amino)hexanoicacid (Compound 9, 6.9 g, 1.0 equiv), Na₂CO₃ (6.83 g, 20 eq), water (56mL), and MeOH (32mL) in respective order. The reaction was aged at roomtemperature for 16h, concentrated to residue, redissolved in water(50mL), and purified on Combiflash C18 gold reverse column (415 g),eluting with water/MeCN. After concentration under vacuum, the productwas dissolved in minimum amount of water, and lyophilized to obtain(S)-2,6-bis(bis((1-(2-(2-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-y0oxy)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)methyl)amino)hexanoicacid (Compound 10) as a white solid. MS(m/z): 1657 [M+Na]⁺

¹HNMR(D₂O, 500 MHz, ppm): 8.05 (s, 2H), 7.91 (s, 2H), 4.62 (t, J=5.0 Hz,4H), 4.57 (t, J=5.0 Hz, 4H), 4.45-4.41 (d, J=8.6 Hz, 4H), 3.99-3.82 (m,28H), 3.80-3.61 (m, 28H), 3.14 (t, J=7.1 Hz, 1H), 2.52 (broad s, 2H),1.99 (s, 6H), 1.98 (s, 6H), 1.73 (m, 2H), 1.60 (m, 2H), 1.29 (m, 2H).

Examples 3-5 Synthesis of TetraGalNAc Ligand Compounds 17a, 17b and 17c

The following Scheme 2 was used to prepare TetraGalNAc Compounds 17a,17b and 17c.

Synthesis of Compound 13

To a solution of 5-chloro-1-pentanol (3.0 g, 24.47 mmol) Compound 11 inDMF (20 mL) was added sodium azide (1.909 g, 29.4 mmol) Compound 12.After being stirred at 60° C. for overnight, the reaction mixture wasconcentrated in vacu. The residue was purified by silica gelchromatography (EtOAc/Hexane 1:3), to give product Compound 13 as clearliquid. ¹H NMR (500 MHz, CDCl₃) δ 3.62 (m, 2H), 3.25 (t, J=6.9 Hz, 2H),1.63-1.53 (m, 4H), 1.45-1.40 (m, 2H).

Synthesis of Compound 15

Compound 13 (0.796 g, 6.16 mmol) and D-galactosamine pentaacetate (2.00g, 5.14 mmol) Compound 14 were suspended in 20 mL DCM, followed byaddition of trifluoromethanesulfonic acid (0.154 g, 1.027 mmol). Theresulting mixture was brought to reflux for overnight. LC-MS indicatedcompleted conversion of SM, the reaction mixture was diluted with EtOAcand washed with sodium bicarbonate and dried over sodium sulfate.Solvent was removed and the residue was purified by ISCO DCM/MeOH from100/0 to 90/10 over 30 min to afford Compound 15 as a white solid. ¹HNMR (500 MHz, CDCl₃) 6: 1.97 (6 H, s), 2.02 (6 H, s), 2.06 (6 H, s),2.15 (6 H, s), 3.28 (6 H, t, J=6.89 Hz), 3.50 (3 H, dt, J=9.63, 6.66Hz), 3.68 (1 H, q, J=5.98 Hz), 3.94-3.92 (7 H, m), 4.16-4.15 (5 H, m),4.73 (2 H, d, J=8.34 Hz), 5.31 (2 H, dd, J=11.16, 3.48 Hz), 5.40-5.38 (5H, m). Calculated mass: [M+H]⁺:C₁₉H₃₁N₄O₉, 459.2; observed: 459.4.

Synthesis of Compound 16.

Lys-alkyne Compound 1 (130 mg, 0.436 mmol) and GalNAc Azide 6 (999 mg,2.178 mmol) were dissolved in THF (5 mL, degassed). Copper (I)bromide-dimethyl sulfide complex (17.91 mg, 0.087 mmol) was added in oneportion to the reaction mixture and the THF solution was stirred forovernight at 40° C. The reaction color changed to blue/green, indicatingCu^(e), fresh sodium ascorbate 37 mg in 0.2 mL of water was added toreaction mixture and allowed to react overnight. The reaction wasconcentrated and purified by RP HPLC 5-60 MeCN(0.5% TFA)/Water(0.5% TFA)over 20 min. The collected fractions were combined and lyophilized toafford Compound 8 as a white solid. Calculated mass:[M+3H]³⁺:C₉₄H₁₄₅N₁₈O₃₈, 2134.0, m/z=711.3; observed: 711.9.

Synthesis of Compound 17a (Ex. 3)

To protected TetraGalNAc Compound 8 (300 mg, 0.141 mmol) in DCM/MeOH=1/15 mL at 0° C. was added Sodium Methoxide (91 mg, 1.688 mmol). Thereaction was stirred for 1 h and quenched by addition of 2 mL of water.Volatile solvent was removed, and the reaction mixture was purified byP4 bio gel with water and the collect fractions were combined andlyophilized to afford Compound 9 as a white solid. Calculated mass:[M+3H]⁺:C₇₀H₁₂₁N₁₈O₂₆, 1629.9, m/z=543.3; observed: 543.8; [M+2H]²⁺:C₇₀H₁₂₀N₁₈O₂₆, 1628.9, m/z=814.5; observed: 814.9.

Synthesis of Compounds 17b and 17c (Ex. 4 and Ex. 5)

Syntheses of Compounds 17b and 17c which have the following structureswere accomplished in a manner similar to that used for Compound 17ausing the appropriate azide source.

Example 6 Scheme of Conjugation of TetraGalNAc Ligands

Scheme 3 below shows a general scheme that can be used to preparetetraGalNAc-siRNA conjugates.

Using the general scheme 3, Conjugates 10-1, 10-2, 10-3, 10a-1, 17a-1,17b-1, 17c-1 can be obtained. The coupling procedure can be performed ona preformed siRNA duplex or on a single strand followed by annealing.Alternatively, one can utilize the protocol outlined in Biocon/ug Chem.2011, 22, pp. 1723-8.

Example 7 Synthesis of TetraGalNAc-siRNA Conjugate 10-1 via TetraGalNAcAcetate Compound 9

To a solution of tetraGalNAc acetate (Compound 9, 58.7 mg, 0.027 mmol)in acetonitrile (1.5 ml) was added DIPEA (2.2 mg, 0.055 mmol) and HATU(10.44 mg, 0.027 mmol). The mixture was stirred at room temperature for30 min, transferred into a solution of siRNA (51) (0.014 mmol) in water(1.5 ml) and acetonitrile (1.5 ml) via a syringe pump over 20 min, andstirred for 30 min before it was concentrated under vacuum down to 1.5mL. Sodium carbonate (218 mg, 2.059 mmol) was then added, followed byMeOH (0.50 ml). The resulted solution was stirred at room temperaturefor 16h, concentrated, purified via dialysis, and lyophilized to yieldtetraGalNAc-siRNA Conjugate 10-1.

Example 8 Synthesis of 3′5′ Bis TetraGalNAc-siRNA Conjugate SingleStrand 18

To a solution of tetraGalNAc acid Compound 10 (41.2 mg, 0.025 mmol) inDMSO (200 uL) was added HATU (9.6mg, 0.025 mmol) and DIPEA (17.6 uL,0.126 mmol). The mixture was stirred at room temperature for 15 min,transferred into a solution of diamino-siRNA (18.8 mg, 2.52 umol) inwater (40 uL) and DMSO (360 uL) and stirred for 30 min. The mixture wasdiluted with water (1.5 mL) and purified on a XBridge Prep Phenyl column(5 uM, 19×250 mm) using a gradient of 0-30% CH₃CN/water containing 100mM TEAA. The fractions were concentrated via dialysis and lyophilized toyield Compound 18.

Example 9 Synthesis of 3′5′ Bis TetraGalNAc-siRNA Duplex Conjugate 19-1

Scheme 4 below was used to prepare TetraGalNAc-siRNA Conjugate 19-1.

A solution of 3′5′ bis tetraGalNAc-siRNA conjugate 18 (13.7 mg, 1.29umol) in water (200 uL) was added to a solution of Guide siRNA (9.3 mg,1.35 umol) dissolved in water (100 uL) and heated at 90 C for 1 minute.The resulting solution was cooled and lyophilized to yield duplex 19-1.

Example 10 Synthesis of TetraGalNAc Ligand Compound 24

The following Scheme 5 was used to prepare tetraGalNAc ligand Compound24.

Synthesis of Compound 22

To a solution of N-BOC-1,3-diaminopropane (Compound 20, 115 mg, 0.660mmol) in 1:1 CH₂Cl₂/CH₃CN (1 mL) at 0° C. was added a solution of3-maleimidopropionic acid N-hydroxysuccinimide ester (Compound 21, 185mg, 0.695 mmol) dissolved in acetonitrile (4 mL) and CH₂Cl₂ (1 mL). Themixture was stirred for 1 h and concentrated in vacuo. The residue waspurified by silica gel chromatography (0-5% MeOH/CH₂Cl₂ to give productCompound 22. Calculated mass: [M+H]⁺:C₁₅H₂₄N₃O₅, 326.2; observed: 326.3.

Synthesis of Compound 23

To a solution of maleimide Compound 22 (56 mg, 0.172 mmol) in CH₂Cl₂ (1ml) was added a solution of 4M HCl (1 ml, 4.00 mmol) in dioxane. Themixture was stirred for 1 h and concentrated in vacuo. The residue wasazeotroped with CH₂Cl₂ (2×) and dried under vacuum to give productCompound 23. Calculated mass: [M+H]⁺:C₁₀H₁₆N₃O₃, 226.1; observed: 226.3.

Synthesis of tetraGalNAc maleimide Compound 24 (Ex. 10)

To a solution of tetraGalNAc acid Compound 10 (100 mg, 0.061 mmol) inDMF (500 uL) was added HATU (34.9 mg, 0.092 mmol), Et₃N (42.6 uL, 0.306mmol) andN-(3-aminopropyl)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamidehydrochloride (16.0 mg, 0.061 mmol). The mixture was stirred at roomtemperature for 1.5 h, acidified with TFA and purified by reverse phase0-50% CH₃CN/water containing 0.1% TFA. The fractions were lyophilized toyield Compound 24. Calculated mass: [M+2H]²⁺: C₇₆H₁₂₅N₂₁O₃₂, 1843.8,m/z=921.9; observed: 922.7.

Example 11 Synthesis of Compound 26

Scheme 6 below was used to prepare Compound 26.

To a degassed solution of 2′-3,17 propargyl siRNA (RNA 25, 33 mg, 4.49umol) and PEG9 SPDP azide (26 mg, 36 umol, prepared from commercialPEG-azide and pyridyl disulfide reagents) in 3:1 DMA/ water (1 mL) wasadded a degassed solution of Copper (I) Bromide-Dimethylsulfide Complex(1.8 mg, 9.0 umol). The mixture was stirred for 72 h at roomtemperature, diluted with water (2 mL), filtered using a 0.45 uM syringefilter and concentrated by dialysis. The concentrated mixture waspurified on a XBridge Prep Phenyl column (5 uM, 19×250 mm) using agradient of 0 — 50% CH₃CN/water containing 100 mM TEAA. The fractionswere concentrated via dialysis and lyophilized to yield Compound 26.

Examples 12-13 Synthesis of Compounds 27 and 28

Scheme 7 below was used to prepare Compounds 27 and 28.

Synthesis of Compound 27 (Ex. 12)

To a solution of 2′-3,17 click PEGS SPDP Conjugate 26 (13.2 mg, 1.50μmol) in water (1 mL) was added a solution of TCEP hydrochloride (9.15mg, 32.2 umol) dissolved in water (0.5 mL). The mixture was stirred atRT for 30 min then purified on a XBridge Prep Phenyl column (5 uM,19×250 mm) using a gradient of 5-40% CH₃CN/water containing 100 mM TEAA.The fractions were concentrated via dialysis and lyophilized to yieldCompound 27.

Synthesis of Compound 28 (Ex. 13)

To a solution of 2′-3,17-click PEG9SH 27 (3 mg, 0.35 μmol) in pH 6.0acetate buffer (100 uL) was added a solution of tetra GalNAc maleimide(5.1 mg, 2.77 μmol) dissolved in pH 6.0 acetate buffer (100 uL). Themixture was stirred at room temperature for 30 min then purified on aXBridge Prep Phenyl column (5 uM, 19×250 mm) using a gradient of 5-40%CH₃CN/water containing 100 mM TEAA. The fractions were concentrated viadialysis and lyophilized to yield Compound 28.

Example 14 Synthesis of 2′-3,17 Bis TetraGalNAc-siRNA Duplex Conjugate29

The procedure detailed for Conjugate 19 was used to duplex 28 to makeConjugate 29.

Example 15 Synthesis of TetraGalNAc Thiol Compound 31

Scheme 8 below was used to prepare Compound 31.

To a solution of tetraGalNAc acid Compound 10 (54 mg, 0.033 mmol) inN,N-dimethylacetamide (500 μl), was added cystamine dihydrochloride 30(14.9 mg, 0.066 mmol), EDC (12.7 mg, 0.066 mmol), HOAT (10.2 mg, 0.066mmol) and DIPEA (57.7 0.330 mmol). The mixture was stirred at roomtemperature for 18 h, then added a solution of DTT (50.9 mg, 0.330 mmol)in N,N-dimethylacetamide (100 μl). The mixture was stirred at roomtemperature for 0.5 h, acidified with TFA and purified by reverse phase0-30% CH₃CN/water containing 0.1% TFA. The fractions were lyophilized toyield Compound 31. Calculated mass: [M+2H]²⁺:C₆₈H₁₁₅N₁₉O₂₉S, 1695.8,m/z=847.9; observed: 848.0.

Examples 16-18 Synthesis of Conjugates 35-37

Scheme 9 below was used to prepare Conjugates 35-37.

Synthesis of Compound 33

To a degassed solution of 2′-click 15 GS Compound 32 (130 mg, 0.019mmol) and (9H-fluoren-9-yl)methyl (2-azidoethyl)carbamate (29.1 mg,0.095 mmol) in 3:1 DMA/water (2 mL) was added a solution of Copper (I)bromide-dimethylsulfide Complex (9.72 mg, 0.042 mmol) dissolved indegassed DMSO (0.32 mL). The mixture was stirred at 45° C. for 2 h,cooled to room temperature, and added pH 8 EDTA (0.5 M, 2 mL) to quenchreaction. Stirred for 15 min and purified on a XBridge Prep Phenylcolumn (5 uM, 30×150 mm) using a gradient of 0-45% CH₃CN/watercontaining 100 mM TEAA. The fractions were concentrated via dialysis. Tothe combined material in water (3 mL) was added a solution of piperidine(936 μL, 1.891 mmol). The mixture was stored at 4° C. for 18 h, dilutedwith water (10 mL) and filtered off solids through syringe filter. AddedpH 8 EDTA (0.5 M, 2 mL), concentrated via dialysis and lyophilized toyield Compound 33.

Synthesis of Compound 34

To a solution of 2′-15 click C2 NH2 GS Compound 33 (43.6 mg, 6.26 μmol)in 200 mM NaHCO3 soln (2000 μl) and formamide (1000 uL) was added asolution of N-Succinimidyl-3-[2-pyridyldithio]propionate (17.9 mg, 0.057mmol) dissolved in DMSO (298 uL). The mixture was stirred at 10° C. for15 min, diluted with water (10 mL) and Formamide (1 mL), andconcentrated by dialysis. Added 2M TEAA (200 uL) and purified on aXBridge Prep Phenyl column (5 uM, 19×250 mm) using a gradient of 5-40%CH₃CN/water containing 100 mM TEAA. The fractions were concentrated viadialysis and lyophilized to yield Compound 34.

Synthesis of 2′-15 TetraGalNAc-siRNA Conjugate 35 (Ex. 16)

To a solution of 2′-15 click C2 NH2 NHS SPDP GS Compound 34 (13 mg, 1.82μmol) in 1:1 formamide/water (200 μl) was added a solution oftetraGalNAc SH (4.62 mg, 2.72 μmol) in formamide (200 uL). The mixturewas stirred at room temperature for 3.5 h, added 2M TEAA (50 uL) andpurified on a XBridge Prep Phenyl column (5 uM, 19×250 mm) using agradient of 2-35% CH₃CN/water containing 100 mM TEAA. The fractions wereconcentrated via dialysis and lyophilized. The resulting solid waspurified on a Proteomix SAX-NP10 column (22.1×50 mm) using a gradient of2-30% (Solvent A: 60:40 TFE/water with 40 mM Et3N, Solvent B: 60:40TFE/water with 40 mM Et3N, 1M Guanidine HCl). The fractions wereconcentrated via dialysis and lyophilized to yield Conjugate 35.

Synthesis of Conjugates 36 and 37 (Ex. 17 and Ex. 18)

The procedure detailed for Conjugate 19-1 was used to duplex Conjugate35 and the appropriate passenger strand to prepare Conjugates 36 and 37,respectively.

Examples 19-26 Synthesis of Conjugates 38-45 (Exs. 19-26)

Schemes 10 and 11 below were used to prepare Conjugates 38-44.

Scheme 11. Examples of different linkers from Table 2 used to conjugatetetraGalNAc to siRNA.Step 1: Passenger-RNA and Linker, Example with Proline to IllustrateProtocol

To a solution of FMOC-PRO-OH (11.11 mg, 0.033 μmol) in 120 μL DMSO wereadded DIPEA (43.2 μl, 0.247 μmol) followed by HATU (10.96 mg, 0.029μmol). The mixture, slightly yellow, was stirred at room temperature for30 min. The mixture was then added to a solution of the oligonucleotidepassenger strand TEAA salt (60 mg, 8.24 μmol) in 500 μL of (10%H₂O/DMSO), and the mixture continued to stir at room temperature for onehour. The reaction mixture showed desired product via LC-MS. To thereaction mixture was added diethylamine (43.0 μl, 0.412 μmol) and themixture was stirred for one hour, confirmed desired product via LC-MS.The reaction mixture was purified by centrifugal dialysis using 3 kDacut-off membrane. The process was repeated three times with water (14 mLeach time). The resulting solution was concentrated, frozen, andlyophilized overnight to yield product as a white fluffy solid. LC/MSconfirms product [7384.9]

Step 2: TetraGalNAc-linker-passenger RNA

To a solution of TetraGalNAc Compound 10 (53.2 mg, 0.033 μmol) in 532 μLDMSO were added DIPEA (42.6 μl, 0.244 μmol) followed by HATU (12.36 mg,0.033 μmol). The mixture, slightly yellow, was stirred at RT for 30 minThe mixture was then added to a solution of the linker-oligonucleotidepassenger strand in 500 μL of DMSO, and the mixture continued to stir atroom temperature for two hours. LC/MS showed desired product. Thereaction mixture was subjected to centrifugal dialysis using 3 kDacut-off membrane. The process was repeated three times with water (14 mLeach time). The resulting solution was purified by Gilson PLC 2020 usingXBRIDGE PHENYL, 10-27% CH₃CN with 200 μM TEAA for 35 minutes. Collectionsolution was concentrated via centrifugal dialysis using 3 kDa cut-offmembrane. The resulting concentrated solution was treated with 1.0N NaCland centrifugal dialysis. The process was repeated five times with water(14 mL each time). The resulting concentrated solution (˜1.5 mL) wasfrozen and lyophilized overnight to yield product as a white fluffysolid. LC/MS confirms product [9002.5].

Step 3: Duplex Formation

To a TetraGalNAc-linker-RNA (18.5 mg, 2.055 μmol) in 1.5 mL of water wasduplexed with ApoB guide strand (14.12 mg, 2.055 μmol) in 1.5 mL ofwater. The mixture was heated at 90° C. for 5 min with stir bar. Theduplex was cooled and stir bar removed. The solution was lyophilizedover two days to yield desired duplex Conjugate 38 as a white fluffysolid. LC/MS confirms product [16048].

ALL the remaining conjugates were prepared using the same generalprocedure.

Examples 27-29 Synthesis of Compounds/Conjugates 46-48

Schemes 12 below was used to prepare Compounds and/or Conjugates 46-48.

Synthesis of RNA Compound 46 (Ex. 27)

SPDP Acid (2.2 mg, 10.3 μmol) was dissolved DMSO 100 μL andN,N-diisopropylethylamine (14.0 μl, 0.08 mmol), HATU (19.6 mg, 0.051mmol) were added sequentially. RNA (15 mg, 2.06 μmol) in 200 μL ofDMSO:Water (9:1) was added and the resulting reaction mixture wasstirred for 1 h, reaction was quenched by addition of 3 mL water anddialyzed down to 500 μL, diluted by formamide to 3 mL and purified bySAX (Buffer A: 60% TFE in water, 20 mM TEA, Buffer B: 60% TFE in water,20 mM TEA, 1 M CsCl, gradient A/B from 100/0 to 35/65 over 15 min). Thecollected fractions were combined and dialyzed against water andlyophilized to afford Compound 46 as a white solid. Calculated mass:[M-H]⁻:C₂₃₄H₃₀₀F₀₈N₇₂O₁₅₀P₂₃S₃, 7480.1; observed: 7483.0.

Synthesis of Conjugate 47 (Ex. 28)

RNA Compound 46 (22 mg, 2.9 μmol) and tetraGalNAc Thiol Compound 31(10.0 mg, 5.9 μmol) were dissolved in formamide:pH=6.8 Tris buffer (3:1)400 μL and stirred for 1 h. The reaction mixture was purified by SAX(Buffer A: 60% TFE in water, 20 mM TEA, Buffer B: 60% TFE in water, 20mM TEA, 1 M CsCl, gradient A/B from 100/0 to 35/65 over 15 min). Thecollected fractions were combined and dialyzed against water andlyophilized to afford Conjugate 47 as a white solid. Calculated mass:[M-H]⁻: C₂₉₇H₄₁₀F₈N₉₀O₁₇₉P₂₃S₃, 9063.9; observed: 9066.2.

Synthesis of Conjugate 48 (Ex. 29)

Conjugate 47 (10.9 mg, 1.20 μmol) and guide strand (7.81 mg, 1.14 μmol)were mixed in RNAse free water 1 mL for 2 h. The reaction mixture waslyophilized to afford duplex Conjugate 48 in quantitative yield.

Examples 30-32 Synthesis of Compounds/Conjugates 49-51

Schemes 13 below was used to prepare Compounds and/or Conjugates 49-51.

Synthesis of RNA Compound 49 (Ex. 30)

33.3 mg of siRNA passenger strand was weighed into a 4mL vial then 1 mL100 mM NaHCO3 was added to dissolve. Added 0.86 uL of propionicanhydride and let stir at RT. After aging ˜2 h, spin dialyzed 3× againstwater. Filtered through frit and the solution was dried vialypophilization to afford 30.8 mg RNA Compound 49.

Synthesis of Conjugate 50 (Ex. 31)

Step 1. Charge 2.8 mg azide, 25.7 mg siRNA, 25 ml N2 sparged DMSO and 4ml water to 40 mL vial. Sparge with N₂. Charge 2.98 mL of Cu/ligandsolution (N₂ sparged, 20/100 umol in 10 ml DMSO). Agitate at RT undersparged N₂.Step 2. Charge Compound 10 and 1 ml DMSO. Charge 6 uL of DIPEA andagitate for 2 min. Charge 6 mg HBTU and agitate for 2 min. Charge siRNAmixture from Step 1. The reaction was not complete so repeated with halfof previous reagent charge. Evaporated the reaction mixture, dialyzedand HPLC purified (X-Bridge Phenyl, TEAA/ACN gradient). Evaporated,dialyze and lyophilized to afford Conjugate 50.

Synthesis of Conjugate 51 (Ex. 32)

Dissolve GS (Conjugate 50) 10.65 mg in 1 ml water and dissolve PS(Conjugate 49) 10.20 mg in 1.17 ml water. Added 8.7 mg of Conjugate 49to all of Conjugate 50 to form a 1:1 duplex. Heat to 900 C for 1 min,cool to RT over 15 min. The solution was filtered and dried vialyophilizaiton to afford Conjugate 51 as a white solid.

RNA Silencing Activity of Compounds Transfected with Lipofectamine inLuciferase Constructs

HEK293 cells stably transfected with luciferase vector that containstarget sites for siRNA in 3′UTR of renilla luciferase were generated.These cells were seeded on 96-well tissue culture plates (Corning:#3903) at a density of 7.5e3 cells per well in DMEM 10% serum media.Cellular plates were then incubated at 37° C./5% CO2 for 24 hr. Afterincubation, plates were treated with test compounds co-transfected withtransfection reagent Lipofectamine 2000 (invitrogen: #11668-019) inOpti-MEM (Gibco: #31985) in accordance to manufacturers protocol. Thetreatment concentrations ranged from 10 nM to 0.03 pM. Treated plateswere then incubated for 24 hr at 37° C./5% CO2. Following treatmentincubation, cells were lysed and processed in accordance to Dual-Glo™Luciferase Assay (Promega: E2920) and read on a TECAN safire2 platereader.

RNA Silencing Activity of Compounds Transfected with Lipofectamine inHepG2 Cells

HepG2 cells (ATCC: HB-8065) were seeded on collagen coated plates(BioCoat: 356649) at a density of 7.5e3 cells per well in DMEM 10% serummedia. Cellular plates were then incubated at 37° C./5% CO2 for 24 hr.After incubation, plates were treated with test compounds co-transfectedwith transfection reagent Lipofectamine 2000 (invitrogen: 11668-019) inOpti-MEM (Gibco: 31985) in accordance to invitrogen protocol. Thetreatment concentrations ranged from 10 nM to 0.03 pM. Treated plateswere then incubated for 24 hr at 37° C./5% CO2. Following treatmentincubation, cells were lysed with PLA Buffer (AB: 4448542) in accordanceto supplied protocol. Resulting cell lysate was reverse transcribed tocDNA using High Capacity cDNA Kit (AB: 4368813) and run through qPCRusing Life Technology 7900.

In vivo Evaluation of RNAi Activity

CD1 female mice were dosed by subcutaneous injection in 200 ul volume.Animals were observed for behavioral or physiological changes. Animalswere sacrificed 72 hrs post dose by CO2 asphyxiation followed byex-sanguination via cardiac puncture. The liver samples were as 3 mmpunches from the medial lobe and put into RNAlater tubes for isolationof total RNA. The mRNA knockdown analysis was conducted by Taqmananalysis using standard procedures.

A summary of in vitro and in vivo data of selected Compounds/Conjugatesis shown in Table 4 presented earlier.

One skilled in the art would readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. The methodsand compositions described herein, as presently representative ofpreferred embodiments, are exemplary and are not intended as limitationson the scope of the invention. Changes therein and other uses will occurto those skilled in the art, which are encompassed within the spirit ofthe invention, are defined by the scope of the claims.

1. A modular composition comprising 1) a single stranded or doublestranded oligonucleotide; 2) one or more tetraGalNAc ligands of Formula(I), (II) or (III), which may be the same or different:

wherein X is —O—, —S—, —CR¹R²— or —NR¹—, wherein R¹ and R² are eachindependently selected from the group consisting of hydrogen andC1-C6alkyl; n is 1 , 2, 3, or 4; and the bond with “

” indicates the point of attachment; optionally, 3) one or more linkers,which may be the same or different; and optionally, 4) one or moretargeting ligands, solubilizing agents, pharmacokinetics enhancingagents, lipids, and/or masking agents.
 2. The modular composition ofclaim 1 comprising: 1) a single stranded or double strandedoligonucleotide; 2) 1-8 tetraGalNAc ligands of Formula (II), which maybe the same or different, wherein X is —O—, —S—, —H₂— or —NH—; and n is1, 2, 3, or 4; 3) 1-16 linkers, which may be the same or different; andoptionally, 4) 1-8 targeting ligands, solubilizing agents,pharmacokinetics enhancing agents, lipids, and/or masking agents.
 3. Amodular composition comprising: 1) a single stranded or double strandedsiRNA; 2) 1-8 tetraGalNAc ligands of Formula (I), (II) or (III), whichmay be the same or different, wherein X is —O—, —S—, —R¹R²— or —NR¹—,wherein R¹ and R² are each independently selected from the groupconsisting of hydrogen and C1-C6alky; and n is 1, 2, 3, or 4; 3) 1-16linkers, which may be the same or different; and optionally, 4) 1-8targeting ligands, solubilizing agents, pharmacokinetics enhancingagents, lipids, and/or masking agents.
 4. The modular composition ofclaim 3, wherein the tetraGalNAc ligands are attached to the siRNA atdifferent 2′-positions of the ribose rings and/or at different terminal3′ and/or 5′-positions of the siRNA; and wherein the tetraGalNAc ligandsare attached to the siRNA optionally via linkers.
 5. The modularcomposition of claim 3, wherein X of Formula (II) is —O—, —S— or —CH₂—;and n is 1,2 or
 3. 6. The modular composition of claim 3, wherein thecomposition comprises 1-4 tetraGalNAc ligands, which may be the same ordifferent.
 7. The modular composition of claim 3, wherein the siRNA isdouble stranded; and wherein the tetraGalNAc ligands are attached to theguide strand or the passenger strand of the siRNA at different2′-positions of the ribose rings of the siRNA.
 8. The modularcomposition of claim 3, wherein the siRNA is double stranded; andwherein the tetraGalNAc ligands are attached to the guide strand or thepassenger strand of the siRNA at different terminal 3′ and/or5′-positions.
 9. The modular composition of claim 3, wherein the siRNAis double stranded; and wherein the tetraGalNAc ligands are attached toboth the guide strand and the passenger strand of the siRNA at different2′-positions of the ribose rings and/or different terminal 3′ and/or5′-positions.
 10. The modular composition of claim 3, wherein thecomposition comprises 2-8 tetraGalNAc ligands of Formula (II), which maybe the same or different; and the tetraGalNAc ligands are attached tothe same strand of the siRNA.
 11. The modular composition of claim 3,wherein the composition comprises 2-8 tetraGalNAc ligands of Formula(II), which may be the same or different; and the tetraGalNAc ligandsare attached to different strands of the siRNA.
 12. The modularcomposition of claim 3, wherein the tetraGalNAc ligands are attached tothe same or different strands of the siRNA via linkers.
 13. The modularcomposition of claim 12, wherein each linker is independently selectedfrom Table
 1. 14. The modular composition of claim 12, wherein eachlinker is independently selected from Table
 2. 15. The modularcomposition of claim 3, wherein the siRNA is double stranded; andwherein the optional targeting ligands, solubilizing agents,pharmacokinetics enhancing agents, lipids, and/or masking agents areattached to the same strand or different strands of the siRNA vialinkers.
 16. A modular composition comprising 1) a double strandedsiRNA; 2) 1-8 tetraGalNAc ligands of Formula (IV), (V) or (VI):

3) 1-16 linkers independently selected from Table 1 , which may be thesame or different; and optionally, 4) 1-8 targeting ligands,solubilizing agents, pharmacokinetics enhancing agents, lipids, and/ormasking agents.
 17. The modular composition of claim 16 comprising: 1) adouble stranded siRNA; 2) 1-3 tetraGalNAc ligands of Formula (V); 3) 1-6 linkers independently selected from Table 1, which may be the same ordifferent; and optionally, 4) 1-3 targeting ligands, solubilizingagents, pharmacokinetics enhancing agents, lipids, and/or maskingagents; wherein the tetraGalNAc ligands are attached to the siRNA atdifferent 2′-positions of the ribose rings and/or at different terminal3′ and/or 5′-positions of the siRNA; and wherein the tetraGalNAc ligandsare attached to the siRNA optionally via linkers.
 18. The modularcomposition of claim 17 comprising 2-3 tetraGalNAc ligands, wherein thetetraGalNAc ligands are attached to the same strand or different strandsof the siRNA via linkers.
 19. The modular composition of claim 17comprising: 1) a double stranded siRNA; 2) 1 -2 tetraGalNAc ligands ofFormula (V); 3) 1-4 linkers independently selected from Table 2, whichmay be the same or different; and optionally, 4) 1-2 targeting ligands,solubilizing agents, pharmacokinetics enhancing agents, lipids, and/ormasking agents; wherein the tetraGalNAc ligands are attached to thesiRNA at different 2′-positions of the ribose rings and/or at differentterminal 3′ and/or 5′-positions of the siRNA; and wherein thetetraGalNAc ligands are attached to the siRNA via linkers.
 20. Themodular composition of claim 19 comprising 2 tetraGalNAc ligands,wherein the tetraGalNAc ligands are attached to the same strand ordifferent strands of the siRNA via linkers.
 21. The modular compositionof claim 19 comprising 1 tetraGalNAc ligand, wherein the tetraGalNAcligand is attached to the siRNA via a linker.
 22. A pharmaceuticalcomposition comprising the modular composition of claim 1 and apharmaceutically acceptable excipient.